The incidence of human malignancies increases significantly with age, suggesting a mechanistic connection between aging (longevity) and carcinogenesis. One aspect of that connection is impaired mitochondrial function, which is observed in both aging cells and cancer cells as aberrant oxidative metabolism. Sirtuin family genes regulate longevity in yeast, C. elegans, and D. melanogaster, and in mammals, three of the seven sirtuin genes are localized to the mitochondria, including SIRT3. These observations led us to hypothesize that SIRT3 might be a tumor suppressor that protects against carcinogenesis by maintaining mitochondrial integrity and efficient oxidative metabolism. The current work demonstrates that the loss of function of SIRT3 results in a cellular environment permissive for carcinogenesis and characterized by aberrant oxidative metabolism. The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3-/- mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3-/- MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3-/- MEFs and reverses the tumor permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3-/- mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondrially localized tumor suppressor.